The primary objective of this proposal is the characterization of the mechanism by which arylhydrazinopyrimidines and arylalkylaminopyrimidines inhibit Bacillus subtilis DNA polymerase III (pol III). I will examine the inhibitor-binding sites of wild type pol III and of a mutant pol III (azp-12) specifically resistant to inhibitors bearing a p-hydroxy group. Approaches to the problem include: 1) derivation of structure-activity relationships of modified inhibitors; 2) synthesis and exploitation of site-directed irreversible inhibitors, and 3) selection of pol III mutants specifically resistant to inhibitors bearing substituents other than p-hydroxy. Structure-activity relationships will be derived by the application of linear free energy equations, by nuclear magnetic resonance analysis of nitrogen-15 labelled inhibitors, and by molecular orbital calculations. Irreversible enzyme inhibitors will be employed to determine inhibitor-enzyme stoichiometry, to aid in the isolation of site-specific peptides, to identify amino acids of the inhibitor-binding site, and to analyze the structural alteration in azp-12 pol III. The properties of additional pol III mutants will be explored by the approaches of 1) and 2) above. As a secondary objective, the selectivity of this group of compounds as inhibitors of only pol III of Gram-positive bacteria will be tested, by screening inhibitor analogs for their capacity to inhibit E. coli pol III and DNA polymerases of animal cells.